Three NOS isoforms evolved to function in human health and disease. Despite structural similarities, each NOS has a different catalytic profile that may broaden their biologic function. In NOS a flavin-containing reductase domain transfers NADPH electrons to a heme in the oxygenase domain and this enables NO synthesis. Catalytic regulation is complex because NO binds to the heme before being released from NOS. We developed a kinetic model that incorporates these and other facets. Our kinetic model reveals that unique catalytic profiles of the three NOS are due to differences in the parameters: Ferric heme reduction (kr), ferric heme-NO dissociation (kd), and oxidation rate of the ferrous heme-NO complex (kox). Although NOS reductase domains control kr and oxygenase domains control kd and kox, the mechanisms and structural basis are unclear. We will perform biochemical, mutagenesis, rapid kinetic, and biophysical experiments test hypotheses regarding how and why kr and kox are broadly regulated in NOS. Aim I. Determine how structural features regulate electron transfer in NOS reductase (NOSr) domains. Our new crystal structure of the intact nNOSr guides these studies: Characterize eNOS and nNOS chimeras in which discreet reductase subdomains are swapped. Examine importance of specific FMN module surface contacts. Determine function of FAD-FMN linker domain. Test importance of H-bonding and other interactions of C-terminal extension in controlling flavin reduction. Compare flavin reduction potentials, electron distribution, and FMN shielding eNOSr and nNOSr and in their isolated FMN modules. Determine how FMN binding site structure controls thermodynamics and electron transfer.Aim II. Examine mechanism and structure function of ferrous heme-NO oxidation by 02 (kox). kox is key because it controls the speed of the futile cycle and also generates an uncharacterized N-oxide product that distinct from NO. Determine reaction mechanism, identify reaction intermediates and the immediate N-oxide product. Examine how NOS heme reduction potential influences kox and the reaction mechanism. Investigate role of N proximal heme loop in controlling kox.Aim III. Generate NOS with novel catalytic behaviors through protein engineering. Variant NOS create will possess combinations of kr and kox designed to greatly shift catalysis toward a futile cycle. These will test understanding of kinetic control mechanism and reveal how NOS products and function can change when key kinetic parameters deviate from their natural ranges.